Comparative Efficacy Of 3 Dimensional ( 3 D) Cell Culture Organoids Vs 2 Dimensional ( 2 D) Cell Cultures Vs Experimental Animal Models In Disease Modeling, Drug Development, And Drug Toxicity Testing

Review Article

=~ Comparative Efficacy of 3Dimensional
°Y° (3D) Cell Culture Organoids Vs
2Dimensional (2D) Cell Cultures Vs
Experimental Animal Models In Disease
modeling, Drug development, And Drug
Toxicity Testing

International Journal of Current Research and Review
DOI: http://dx.doi.org/10.31782/IJCRR.2019.11242

IJCRR
Section: Healthcare
Sci. Journal Impact
Factor: 6.1 (2018)
ICV: 90.90 (2018)

Santenna Chenchulat, Sunil Kumart, Shoban Babu V2

‘Department of Pharmacology, Demonstrator, AMS, Bhopal; Department of Pharmacology, Assistant Professor, AIMS, Jodhpur.

ABSTRACT

Historically animal studies and 2D cell culture models have been strengthening biomedical and pharmaceutical research, with
many limitations. Currently, new drug development for many diseases like cancer is an important necessity. An organoid is a
miniaturized version of an organ produced in vitro that shows realistic micro-anatomy, is capable of self-renewal and self-organ-
ization and exhibits similar functionality as the tissue of origin. While their size is small (typically < 3 mm in diameter), organoids
are stable model systems of organs and tissues that are amenable to long-term cultivation and manipulation. They are classified
into those that are tissue-derived and those that are stem cell-derived. They help in both in vivo and in vitro investigation and
represent one of the latest innovations in the research for a model to recapitulate the physiologic processes of whole organisms.
They reduce experimental complexity, and are compliant to real-time imaging techniques, and more importantly, they enable the
study of aspects of human development and disease, drug toxicity in a clear fashion that is not easily or correctly modelled in
animals and 2D cell cultures. However 3D organoids have also had some limitations like vascularity, inflammatory system, etc.
Despite these limitations, it is evident that organoids have great potential to revolutionize the way we approach disease model-
ling, drug discovery, and toxicology.

Key Words: 3D organoids, 2D cell culture, Drug discovery, Organ toxicity, High-throughput screening

INTRODUCTION

Traditionally animal studies using rats and mice etc., and
2D cell culture models have been used over the past decades
in the field of biomedical research. Both these models are
extremely helpful in disease modelling prior to the advent
of the three-dimensional (3D) cell culture organoid technol-
ogy. Nude and severe combined immunodeficiency (SCID)
mice are immunodeficient mice, which are most commonly
used as conferee of human cells or tissues as they accept for-
eign tissues or cells relatively easily due to a lack of host im-
munity. Another variety of rodents are humanized mice; this
variety of mice lacks an innate immune system because these

varieties completely procreated with human immune cells by
hematopoietic stem cell transplantation [1]. Such humanized
mouse models are particularly useful to model disease pa-
thology and to allow for the assessment of potential thera-
peutic candidates. They provide a systemic environment
to study disease pathology due to the presence of an intact
immune system and blood circulation, which are essentially
impossible in 2D models. Many studies have been succeed-
ed in providing new insight into disease pathogenesis using
humanized rodents in modelling human diseases. However,
there are many raising questions on the pertinence of using
mouse models to study human diseases [2]. 2D cell cultures
used to study different cell types, along with drug screening

Corresponding Author:

Phone: 987220348; E-mail: csanten7@gmail.com
ISSN: 2231-2196 (Print)

Received: 12.11.2019 Revised: 02.12.2019

Santenna Chenchula, Department of Pharmacology, Demonstrator, AIIMS, Bhopal.

ISSN: 0975-5241 (Online)

Accepted: 16.12.2019

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Chenchula et.al.: Comparative efficacy of 3dimensional (3D) cell culture organoids vs 2dimensional (2D) cell cultures

and testing. Usually, it contains the monolayer system which
allows cell growth over a polyester or glass flat surface pre-
senting a medium that feeds the growing cell population
[3].Endless biological breakthroughs occurred through 2D
cell culture research(quote few examples). However these
2D cell culture models have limitations due to their sim-
plicity, lack of tissue-specific architecture, mechanical and
biochemical cues, and cell-to-cell and cell-to-matrix interac-
tions, so this model can’t accurately depict and simulate the
rich environment and complex processes observed in vivo
such as cell signalling, chemistry or geometry, which makes
them relatively poor models to predict drug responses for
certain diseases like cancer [4]. As a result, data gathered
with 2D cell culture methods could be non-predictive or mis-
leading. Use of murine animal models for disease modelling,
drug testing, and therapeutic development is not only costly
and time-consuming but may not mimic biological responses
in humans due to species differences. Considering all these
limitations of animal studies and 2D cell culture system
are not effective in disease modelling and drug research.
However 3D cell culture organoids serve to overcome the
challenges faced by 2D culture and animal disease models.
Nonetheless, such modelling of human diseases in 3D cell
culture organoids may still need to be validated in vivo, ulti-
mately using a humanized mouse model.

3Dimensional (3D) cell culture Organoids

An organoid is “a collection of organ-specific cell types
that develops from stem cells or organ progenitors and self-
organizes through cell sorting and spatially restricted line-
age commitment in a manner similar to im vivo.An organoid
is a miniaturized version of an organ produced in vitro that
shows realistic micro-anatomy, is capable of self-renewal
and self-organization and exhibits similar functionality as
the tissue of origin. While their size is small (typically < 3
mm in diameter), organoids are stable model systems of or-
gans and tissues that are amenable to long-term cultivation
and manipulation. 3D cell culture organoid constructs com-
posed of multiple cell types that originate from stem cells
by means of self-organization and is capable of simulating
the architecture and functionality of native organs [5,6].They
constitute a rapidly expanding family of dish-based, 3D de-
veloping tissues that show sensible microanatomy. They
are generated with the use of somatic cells, adult stem cells
(progenitor cells), or pluripotent stem cells. They are similar
to their original organs, so they hold many advantages over
traditional two-dimensional cultures and even animal mod-
els for use in medical research and the development of new
treatments.

3D cell culture Organoids are of two types, tissue and stem
cell organoids, depending on how the organ buds are formed.
Stem cell-derived organoids may only recapitulate the first
few months of development, but not the stages beyond.

Therefore they potentially lack some cell types of interest for
biomedical research. However, tissue organoids generated
from the isolated adult stem or progenitor cells are resected
fragments of organ tissues are more suitable in the field of
biomedical and pharmaceutical research.Till now there are
many in vitro organoids have been established to resemble
various tissues, including functional organoids for thyroid,
pancreas, liver, stomach, intestine, cerebral cortex, pituitary,
etc.[7,8].These 3D cell culture organoid tissues help in both
in vivo and in vitro investigation and represent one of the
latest innovations in the research for a model to recapitu-
late the physiologic processes of whole organisms [9,10,11].
They display near-physiologic cellular composition and be-
haviours. Compared to animal models, 3D organoids will
reduce experimental complexity, and are compliant to real-
time imaging techniques, and, more importantly, they enable
the study of aspects of human development and disease that
are not easily or correctly modelled in animals (Fig 1, Table
1).In contrast to conventional 2D methods, cells cultured in
a 3D cell culture organoids may exhibit unique biochemical
and morphological features, which are similar to their cor-
responding tissues in vivo. Nowadays organoids use in bio-
medical research progressing for the modelling of a disease,
screening of new drugs, and pathophysiological characteri-
zation.Although organoid technology is expanded in the
biomedical field, yet it is still in its infancy. There are many
practical challenges to overcome before it can be widely im-
plemented in disease modeling, drug discovery, and toxico-
logical applications (Table 1). Still, there are many advan-
tages of 3d organoids over animal experimental models and
2D cell culture organoids (Figure 1) (Table 1).

Disease modelling

Disease modelling plays an important role in drug discov-
ery. Animal model studies of both in vitro and in vivo were
commonly employed for disease modelling. 3D cell cultures
Organoids Bridge the gap between 2D cell culture and in
vivo animal model studies. A range of 3D cell cultures has
been applied until now to understand the mechanisms of dif-
ferent diseases.3D cell cultures organoids may provide more
fundamental insights into development, homeostasis, and
pathogenesis and may offer new translational approaches for
the diagnosis and treatment of disease. For example to study
the human brain, develop cerebral organoids to recapitulate
human-specific neurogenic processes [12]. Human cerebral
organoids have been grown in a microfabricated compart-
ment that allows long-term in situ imaging. This system
has been used to model the physics of cortical folding and
to study the mechanism underlying lissencephaly, which is
caused by mutations in LIS1 [13].

Cerebral organoids have also been used to show that the
Zika virus preferentially infects neural progenitors and re-
duces their multiplication and viability, which may, in turn,

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Chenchula et.al.: Comparative efficacy of 3dimensional (3D) cell culture organoids vs 2dimensional (2D) cell cultures

be a cause of Zika virus—associated microcephaly [14]. This
in vitro reflection of in vivo phenotype helps to investigate
disease mechanisms and develop new treatment strategies in
less time, and also reduces experimental animal use.

Recently 3D cell culture technologies generated novel 3D
neural cell culture models that recapitulate Alzheimer’s dis-
ease (AD) pathology including robust AB deposition and
Af-driven NFT-like tau pathology [15]. These novel orga-
noids of AD hold a promise for a novel platform that can
be used for mechanism studies in human brain-like environ-
ment and highly selective drug screening. In the past decade,
AD transgenic mice have been used as a standard preclinical
model for testing candidate AD drug targets. The test com-
pounds are tested in AD transgenic mice with multiple doses
to explore their potential toxicity and the impact on AD pa-
thology, including pathogenic AB accumulation, p-tau accu-
mulation and the behavioural and memory deficits. This pro-
cess takes more than 2-3 years and is relatively expensive.
The only small number of test compounds can pass through
this process, and a majority of Alzheimer drug targets which
showed efficacy in all the biochemical, cell culture and Alz-
heimer disease transgenic models, have failed to show ef-
ficacy in human clinical trials [14]. Even 3D culture models
of Alzheimer disease are relatively cheaper and faster (6—10
weeks for our 3D culture model; 12 weeks for 3D organoid
models) as compared to AD transgenic mouse model [16].
A 3D cell culture organoid technology has integrated with
other technologies, including genome editing, single-cell
genomics, live imaging, and microfluidics, thus provid-
ing new insights into developmental processes and disease
pathogenesis as well as enabling translational approaches to
the diagnosis and treatment of disease.

Neural cortical organoids which are induced from human in-
duced pluripotent stem cells (hiPSCs) obtained from individ-
uals with severe idiopathic autism spectrum disorder (ASD)
are used to model autism. These cortical organoids exhibit a
decreased cell cycle duration, indicative of flustered cell cy-
cle potential, and showed an overproduction of GABAergic
inhibitory neurons, providing critical insight into the patho-
genesis of ASD. The success of cortical organoids in model-
ling autism is largely due to the ability to model embryonic
telencephalic development seen in the third trimester of hu-
man development, as well as recapturing the regulatory net-
works of GABAergic neuron production. The authors identi-
fied gene expression of FOXG/ could potentially be used
as a biomarker of severe autism.Irregularity of FOXG/ gene
predominant in these cortical organoids provides an under-
standing of the alterations in the dynamics of brain growth
and differentiated neurons [17].

Cancer modeling
Treating cancer has been challenging since long back, due
to the complexity and heterogeneity of tumours, leading to

resistance to chemotherapy. This complexity is partly due to
the interaction between the tumor and its microenvironment
[18]. The tumor microenvironment (TME) mainly consists
of different non-cancer cell types and their stroma, such as
fibroblasts, immune cells like lymphocytes and macrophag-
es, mesenchymal cells, and endothelial cells (EC), which all
have a specific role in the physiology, structure, and func-
tion of the tumour. In cancer research, well-established and
characterized 2D cancer cell culture has massively contrib-
uted to the understanding of carcinogenesis from cell pro-
liferation and migration to drug discovery [18].Yet cancer
remains a complex disease that is still not fully understood,
especially due to its close interactions with its surrounding
cells.2D cultures generally fail to translate accurately the
natural in vivo setting. When cells are cultured in 2D, they
grow as monolayers, which lead to polarized cell adhesion
and two-dimensional contact with neighbouring cells. This
physical characteristic allows them to receive a homogene-
ous amount of nutrients and growth factors from the media,
resulting in abnormal cell spreading, an unrealistic distribu-
tion of cell surface receptors, and selection for specific cell
sub-populations best adapted to 2D in vitro growth. There
are some shortcomings in animal models like; slow growth
rates of many engrafted primary cancers in patient-derived
xenografts (PDX) models, and tumour heterogeneity [19].
So all these limitations of 2D cell culture and animal studies
have encouraged the development of many 3D cell culture
methods to better translate the complex pathophysiological
features of the TME in vitro.Emerged3D cell culture orga-
noid models have gained much popularity in studying tumor
biology because standard 2D models are ineffective to an-
swer questions regarding indolent disease, metastatic colo-
nization, dormancy, relapse, and the rapid evolution of drug
resistance. Different types of the Primary tumor organoids
of colon, prostate, breast, ovarian and pancreas are success-
fully developed [20]. These are called “tumouroids”, and
they emerged as preclinical models that have the potential
to predict an individual patient’s response to treatment. Li-
brary of an organoid representing different grades of colo-
rectal tumors revealed a decreased dependence on niche fac-
tors along with the transition from normal tissue to adenoma
to carcinoma [20]. Niche factor dependency is found to be
primarily associated with the genetic makeup of a tumour.
So these tumoroids are a means of linking cancer-related
genomic data to tumour biology and can provide a substrate
for drug screening and personalized treatment.

Drugs discovery

Drug discovery is heavily reliant on high-throughput screen-
ing (HTS); the process of identifying hits by testing a large
number of diverse chemical structures against disease targets
and is characterized by its simplicity, efficacy, low cost per
assay, and high efficiency [21]. 3D cell cultures organoids
have an important role to discover novel mechanisms and

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targets to accelerate lead identification and validation, as
the gene expression patterns found in 3D models are closer
to in vivo, compared to 2D monolayer models [22]. Drugs
identified on the basis of 2D-cultured cell lines give aber-
rant responses, and they have a considerable impact on the
drug selection compared to drugs identified on the 3D cell
culture organoid tissues. For instance, cancer cells grown as
a monolayer have a deregulated cell cycle, often doubling
every 24 h, while tumours in vivo typically show only a few
percents of actively cycling cells and only have a marginally
higher rate of proliferation compared with healthy tissue. As
a result, cancer drugs selected on the basis of arresting pro-
liferation in culture often do little in vivo, or, if they do, will
also show adverse effects in healthy tissue[23].

Drug toxicity testing

Every year billions are spent on developing targets identified
from in vitro systems through to Phase III trials in patients.
The vast majority of these compounds fail due to either un-
acceptable toxicities or limited efficacy in humans. So tradi-
tional 2D cell systems are ineffective in predicting clinical
responses. Organ toxicity is one of the common limitations
of drug development failures and withdrawals after market-
ing [24]. Among all organ toxicities currently, liver, heart,
kidney, and brain toxicity account for more than 70% of drug
attrition and withdrawal from the market. Renal and hepato-
toxicity are the very common leading cause of drug attrition,
is often accompanied by dysfunction in the bile transport sys-
tem. Current toxicology screening tests that use cell lines and
animal models often do not predict complete adverse effects
profile of a new drug in human beings, essentially in whom
renal and hepatic toxicities are among the most common.3D
cell culture Organoids can also be useful in drug screen-
ing applications for assessing efficacy and safety, they are
powerful in assessing drug-induced toxicity. They may offer
more accurate means of toxicity prediction than animal mod-
els. Organ buds of brain, liver, heart, and kidney can be used
to assess drug toxicity [25]. For instance, a brain organoid
which was produced by combining human embryonic stem
cell (ESC) derived neural progenitor cells, endothelial cells,
Mesenchymal stem cells(MSCs), and microglia/macrophage
precursors on chemically defined polyethylene glycol hydro-
gels. Machine learning was used to build a predictive model
from changes in global gene expression when being exposed
to 60 training compounds (34 toxic and 26 nontoxic chemi-
cals) [26]. This model was then used to correctly classify 9
additional chemicals in a blinded trial.

The hepatic bi-progenitor cell line HepaRG is a unique cell
line showing great plasticity, which differentiates to both
canaliculae-like and hepatocyte-like cells. Human liver or-
ganoids obtained using HepaRG cell line; a terminally dif-
ferentiated hepatic cell line derived from a human hepatic
progenitor cell line, and have been shown to produce human-

specific metabolites [27]. This is very useful because gener-
ally, the human liver metabolizes drugs in a manner distinct
from animal liver. Of note, these HepaRG 3D organoid cul-
tures are more sensitive to paracetamol or rosiglitazone-in-
duced toxicity but less sensitive to troglitazone-induced tox-
icity than the 2D cultures. Kidney organ buds from human
iPSC cells were found to differentially apoptosis in response
to cisplatin, a nephrotoxicant, showing such organoids rep-
resent powerful models of the human organ for drug-in-
duced nephrotoxicity. Favourably organoids of the kidney
have been shown to recapitulate the nephrotoxic effects of
cisplatin and gentamicin [28, 29, 30]. Also, organoids have
advantages include their genetic stability and scalability for
high-throughput screens e.g. Like human nephron progenitor
cells have a nearly unlimited ability to self-renew in three-
dimensional culture, which could be beneficial for standardi-
zation of nephrotoxicity screens [5].

Organs-on-chips and other 3D cell culture models were the
food and drug administration (FDA) approved to evaluate
drug-induced toxicity [31]. Recently the FDA has started
testing three-dimensional “liver-on-a-chip” constructs to
screen for the hepatic toxicity of compounds used in food
additives, nutritional supplements, and cosmetics. Heart-
on-a-chip devices were useful for assessing drug-induced
cardiotoxicity. The lung-on-chip model consists of channels
lined by closely apposed layers of human pulmonary epithe-
lial and endothelial cells that experience air and fluid flow,
enabling the detection of drug toxicity-induced pulmonary
oedema observed in human cancer patients treated with inter-
leukin-2 at similar doses and over the same time frame. They
were also found that both angiopoietin-1 and GSK2193874
(a transient receptor potential vanilloid 4 ion channel inhibi-
tor) were effective at preventing the drug toxicity-induced
pulmonary oedema [32].

Cell-based therapy using Intestinal organoids
Cystic fibrosis is an autosomal recessive disease caused
by the cystic fibrosis transmembrane-conductor regulator
(CFTR) gene mutation. Colon organoids are generated from
patients with cystic fibrosis. Genome editing using CRISPR/
Cas9 (clustered regularly interspaced short palindromic re-
peat/associated protein 9) has been used to correct mutations
in CFTR and to restore the functionality of the CFTR protein
in colon organoids derived from patients with cystic fibrosis
[33]. However such studies suggest that organoids may be a
source of cells in future approaches to cell therapy.

Limitations of 3D cell culture organoids

Although organoids have a wide range of potential applica-
tions, the current version still represents a somewhat rough
model, and researchers still grapple with obstacles of this
technology. Firstly the present 3D organoid systems have re-
producibility limitations as there is little or no control over

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Chenchula et.al.: Comparative efficacy of 3dimensional (3D) cell culture organoids vs 2dimensional (2D) cell cultures

how cells self-organize into the organoids [34]. So, these pro-
tocols are unable to produce exact replications of organoid
spheres of the same dimensions (size and shape), cellular
composition, phenotypic and molecular characteristics. The
“Tissues In a Dish” contains only an epithelial layer with-
out native microenvironment including surrounding mesen-
chyme, immune cells, nervous system, or muscular layer .to
overcome this limitation, the stem cell niche was proposed as
a major aspect in which scientists can manipulate to improve
the reproducibility of the organoids obtained

Vascularization is another important limitation of 3D orga-
noids which is important for the supply of oxygen and nutri-
ents to be supplied throughout the mass, encouraging better
development of cells into tissue-like structures and for the
cells that are within the mass to be able to survive and func-
tion as well as cells on the periphery. Even a vascularized
organoid only will be able to capture the drug uptake, circu-
lation, and metabolism that occur in the body [35].

3D organoid models are unable to produce the process of
inflammation that occurs in vivo, which involves a multitude
of cell types (endothelial cells, monocytes, macrophages,
leukocytes) and cellular processes (leukocyte-endothelial
cell adhesion, leukocyte extravasation and transmigration,
and monocyte to macrophage differentiation) [36]. Blood is
very crucial for the body’s inflammatory response by serving
as the carrier medium for immune cells. These immune cells
eventually move to sites of injury through chemo-attraction
and interact with blood vessel walls (through adhesion mol-
ecules presented by endothelial cells) before extravasating to
inflamed loci. Therefore, in order to mimic inflammation in
3D in vitro models, vascularization has to first be established,
blood perfusion has to occur, and immune cell types must be
present. A 3D model that can successfully incorporate such
an inflammation niche will be an extremely powerful tool
for studying atherosclerotic vascular diseases, inflammatory
skin diseases, interstitial nephritis, and even inflammatory
bowel disease. In fact, humanized mouse models provide a
systemic environment to holistically study disease pathology
due to the presence of an intact immune system and blood
circulation, which are essentially impossible in 2D and 3D
models.

DISCUSSION AND CONCLUSION

3D cell culture organoids have been a major advancement
to increase productivity and newer drugs development in
pharmaceutical research and development. They hold great
potential as a tool in new drug discovery—ranging from dis-
ease modelling to drug discovery, drug toxicity testing and
as a new type of therapeutics/replacement therapy that may
transform our lives. But these 3D cell culture organoids also
have some limitations like lack of reproducibility, vascular-

ity and lack of inflammation niche .So in future 3D cell mod-
els have to overcome these challenges and more research de-
velopments are needed in 3D organoid cell models, and will
no doubt bring them closer to reaching these limitations in
the biomedical and pharmaceutical field of research.

ACKNOWLEDGEMENT

Authors acknowledge the immense help received from the
scholars whose articles are cited and included in references
to this manuscript. The authors are also grateful to authors /
editors / publishers of all those articles, journals and books
from where the literature for this article has been reviewed
and discussed.

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Table 1: 3D cell culture organoids vs. 2D cell culture models vs. animal models

Physiologic enactment Semi physiologic
Human development and disease modeling Yes
High-throughput screening Yes

Manageability

Organogenesis modeling

Vascularization and immune system

cost

variability

Good, may have some experimental

Suitable for the study of cell-cell com-

munication, morphogenesis;

No

Less

Limited Physiologic
Poor Yes

Yes No
Excellent Limited
poor Yes

No Yes

Less High

Int J Cur Res Rev | Vol 11 - Issue 24 - December 2019

Chenchula et.al.: Comparative efficacy of 3dimensional (3D) cell culture organoids vs 2dimensional (2D) cell cultures

e fe

i ol

Reprogramming

Tissue stem
cells

x

Figure 1: 3D cell culture organoids vs. 2D cell culture models vs. animal models.

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